Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance.

BACKGROUND: The E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts. RESULTS: Using GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9% cell loss with survival rate of only < 0.03%. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~ 50% of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins. CONCLUSIONS: We demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9% cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.

BioStruct-Africa: empowering Africa-based scientists through structural biology knowledge transfer and mentoring - recent advances and future perspectives.

Being able to visualize biology at the molecular level is essential for our understanding of the world. A structural biology approach reveals the molecular basis of disease processes and can guide the design of new drugs as well as aid in the optimization of existing medicines. However, due to the lack of a synchrotron light source, adequate infrastructure, skilled persons and incentives for scientists in addition to limited financial support, the majority of countries across the African continent do not conduct structural biology research. Nevertheless, with technological advances such as robotic protein crystallization and remote data collection capabilities offered by many synchrotron light sources, X-ray crystallography is now potentially accessible to Africa-based scientists. This leap in technology led to the establishment in 2017 of BioStruct-Africa, a non-profit organization (Swedish corporate ID: 802509-6689) whose core aim is capacity building for African students and researchers in the field of structural biology with a focus on prevalent diseases in the African continent. The team is mainly composed of, but not limited to, a group of structural biologists from the African diaspora. The members of BioStruct-Africa have taken up the mantle to serve as a catalyst in order to facilitate the information and technology transfer to those with the greatest desire and need within Africa. BioStruct-Africa achieves this by organizing workshops onsite at our partner universities and institutions based in Africa, followed by post-hoc online mentoring of participants to ensure sustainable capacity building. The workshops provide a theoretical background on protein crystallography, hands-on practical experience in protein crystallization, crystal harvesting and cryo-cooling, live remote data collection on a synchrotron beamline, but most importantly the links to drive further collaboration through research. Capacity building for Africa-based researchers in structural biology is crucial to win the fight against the neglected tropical diseases, e.g. ascariasis, hookworm, trichuriasis, lymphatic filariasis, active trachoma, loiasis, yellow fever, leprosy, rabies, sleeping sickness, onchocerciasis, schistosomiasis, etc., that constitute significant health, social and economic burdens to the continent. BioStruct-Africa aims to build local and national expertise that will have direct benefits for healthcare within the continent.

The N-acyltransferase Lnt: Structure-function insights from recent simultaneous studies.

Bacterial lipoproteins have been researched for decades due to their roles in a large number of biological functions. There were no structures of their main three membrane processing enzymes, until 2016 for Lgt and LspA, and then 2017 for Lnt with not one but three simultaneous, independent publications. We have analyzed the recent findings for this apolipoprotein N-acyltransferase Lnt, with comparisons between the novel structures, and with soluble nitrilases, to determine the significance of unique features in terms of substrate's recognition and binding mechanism influenced by exclusive residues, two transmembrane helices, and a flexible loop.

Efflux drug transporters at the forefront of antimicrobial resistance.

Bacterial antibiotic resistance is rapidly becoming a major world health consideration. To combat antibiotics, microorganisms employ their pre-existing defence mechanisms that existed long before man's discovery of antibiotics. Bacteria utilise levels of protection that range from gene upregulation, mutations, adaptive resistance, and production of resistant phenotypes (persisters) to communal behaviour, as in swarming and the ultimate defence of a biofilm. A major part of all of these responses involves the use of antibiotic efflux transporters. At the single cell level, it is becoming apparent that the use of efflux pumps is the first line of defence against an antibiotic, as these pumps decrease the intracellular level of antibiotic while the cell activates the various other levels of protection. This frontline of defence involves a coordinated network of efflux transporters. In the future, inhibition of this efflux transporter network, as a target for novel antibiotic therapy, will require the isolation and then biochemical/biophysical characterisation of each pump against all known and new antibiotics. This depth of knowledge is required so that we can fully understand and tackle the mechanisms of developing antimicrobial resistance.

Concentration-dependent effects of acute and chronic neonicotinoid exposure on the behaviour and development of the nematode Caenorhabditis elegans.

BACKGROUND: Neonicotinoid insecticides are under review owing to emerging toxicity to non-target species. Interest has focused on biological pollinators while their effects on other organisms that are key contributors to the ecosystem remain largely unknown. To advance this, we have tested the effects of representatives of three major classes of neonicotinoids, thiacloprid, clothianidin and nitenpyram, on the free-living nematode Caenorhabditis elegans (C. elegans), as a representative of the Nematoda, an ecologically important phylum contributing to biomass. RESULTS: Concentrations that are several-fold higher than those with effects against target species had limited impact on locomotor function. However, increased potency was observed in a mutant with a hyperpermeable cuticle, which shows that drug access limits the effects of the neonicotinoids in C. elegans. Thiacloprid was most potent (EC50 714 µm). In addition, it selectively delayed larval development in wild-type worms at 1 mm. CONCLUSION: C. elegans is less susceptible to neonicotinoids than target species of pest insect. We discuss an approach in which this defined low sensitivity may be exploited by heterologous expression of insect nicotinic acetylcholine receptors from both pest and beneficial insects in transgenic C. elegans with increased cuticle permeability to provide a whole organism assay for species-dependent neonicotinoid effects. © 2017 Society of Chemical Industry.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease from Pseudomonas aeruginosa.

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid-polyamine-organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP from Pseudomonas aeruginosa was recombinantly expressed in Escherichia coli and purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Šresolution. The diffraction data were indexed in space group P21, with unit-cell parameters a = 169.53, b = 169.53, c = 290.13 Å, γ = 120°.

Heterologous expression and purification of an active human TRPV3 ion channel.

The transient receptor potential vanilloid 3 (TRPV3) cation channel is widely expressed in human tissues and has been shown to be activated by mild temperatures or chemical ligands. In spite of great progress in the TRP-channel characterization, very little is known about their structure and interactions with other proteins at the atomic level. This is mainly caused by difficulties in obtaining functionally active samples of high homogeneity. Here, we report on the high-level Escherichia coli expression of the human TRPV3 channel, for which no structural information has been reported to date. We selected a suitable detergent and buffer system using analytical size-exclusion chromatography and a thermal stability assay. We demonstrate that the recombinant purified protein contains high α-helical content and migrates as dimers and tetramers on native PAGE. Furthermore, the purified channel also retains its current inducing activity, as shown by electrophysiology experiments. The ability to produce the TRPV3 channel heterologously will aid future functional and structural studies.

Dual-mode phospholipid regulation of human inward rectifying potassium channels.

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using (86)Rb(+) flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP(2) increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP(2), and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP(2) and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.

Direct and specific activation of human inward rectifier K+ channels by membrane phosphatidylinositol 4,5-bisphosphate.

Many ion channels are modulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)), but studies examining the PIP(2) dependence of channel activity have been limited to cell expression systems, which present difficulties for controlling membrane composition. We have characterized the PIP(2) dependence of purified human Kir2.1 and Kir2.2 activity using (86)Rb(+) flux and patch clamp assays in liposomes of defined composition. We definitively show that these channels are directly activated by PIP(2) and that PIP(2) is absolutely required in the membrane for channel activity. The results provide the first quantitative description of the dependence of eukaryotic Kir channel function on PIP(2) levels in the membrane; Kir2.1 shows measureable activity in as little as 0.01% PIP(2), and open probability increases to ∼0.4 at 1% PIP(2). Activation of Kir2.1 by phosphatidylinositol phosphates is also highly selective for PIP(2); PI, PI(4)P, and PI(5)P do not activate channels, and PI(3,4,5)P(3) causes minimal activity. The PIP(2) dependence of eukaryotic Kir activity is almost exactly opposite that of KirBac1.1, which shows marked inhibition by PIP(2). This raises the interesting hypothesis that PIP(2) activation of eukaryotic channels reflects an evolutionary adaptation of the channel to the appearance of PIP(2) in the eukaryotic cell membrane.

Unusual binding interactions in PDZ domain crystal structures help explain binding mechanisms.

PDZ domains most commonly bind the C-terminus of their protein targets. Typically the C-terminal four residues of the protein target are considered as the binding motif, particularly the C-terminal residue (P0) and third-last residue (P-2) that form the major contacts with the PDZ domain's "binding groove". We solved crystal structures of seven human PDZ domains, including five of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7 show a binding mode with only the C-terminal P0 residue bound in the binding groove. Importantly, in some cases, the P-2 residue formed interactions outside of the binding groove, providing insight into the influence of residues remote from the binding groove on selectivity. In the GRASP structure, we observed both canonical and noncanonical binding in the two molecules present in the asymmetric unit making a direct comparison of these binding modes possible. In addition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allow comparison with canonical binding for the PDLIM PDZ domain family. Although influenced by crystal packing arrangements, the structures nevertheless show that changes in the positions of PDZ domain side-chains and the alpha B helix allow noncanonical binding interactions. These interactions may be indicative of intermediate states between unbound and fully bound PDZ domain and target protein. The noncanonical "perpendicular" binding observed potentially represents the general form of a kinetic intermediate. Comparison with canonical binding suggests that the rearrangement during binding involves both the PDZ domain and its ligand.

Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae.

The inward rectifier family of potassium (KCNJ) channels regulate vital cellular processes including cell volume, electrical excitability, and insulin secretion. Dysfunction of different isoforms have been linked to numerous diseases including Bartter's, Andersen-Tawil, Smith-Magenis Syndromes, Type II diabetes mellitus, and epilepsy, making them important targets for therapeutic intervention. Using a family-based approach, we succeeded in expressing 10 of 11 human KCNJ channels tested in Saccharomyces cerevisiae. GFP-fusion proteins showed that these channels traffic correctly to the plasma-membrane suggesting that the protein is functional. A 2-step purification process can be used to purify the KCNJ channels to >95% purity in a mono-dispersed form. After incorporation into liposomes, (86)Rb(+) flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels.

Regulator of G-protein signaling 14 (RGS14) is a selective H-Ras effector.

BACKGROUND: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Galpha-mediated GTP hydrolysis ("GTPase-accelerating proteins" or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Galpha GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras. METHODOLOGY/PRINCIPAL FINDINGS: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co-transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor-mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling. CONCLUSIONS/SIGNIFICANCE: In cells, RGS14 facilitates the formation of a selective Ras.GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras-binding domain architecture with RGS14.

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.

The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins.

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.

Structure of PICK1 and other PDZ domains obtained with the help of self-binding C-terminal extensions.

PDZ domains are protein-protein interaction modules that generally bind to the C termini of their target proteins. The C-terminal four amino acids of a prospective binding partner of a PDZ domain are typically the determinants of binding specificity. In an effort to determine the structures of a number of PDZ domains we have included appropriate four residue extensions on the C termini of PDZ domain truncation mutants, designed for self-binding. Multiple truncations of each PDZ domain were generated. The four residue extensions, which represent known specificity sequences of the target PDZ domains and cover both class I and II motifs, form intermolecular contacts in the expected manner for the interactions of PDZ domains with protein C termini for both classes. We present the structures of eight unique PDZ domains crystallized using this approach and focus on four which provide information on selectivity (PICK1 and the third PDZ domain of DLG2), binding site flexibility (the third PDZ domain of MPDZ), and peptide-domain interactions (MPDZ 12th PDZ domain). Analysis of our results shows a clear improvement in the chances of obtaining PDZ domain crystals by using this approach compared to similar truncations of the PDZ domains without the C-terminal four residue extensions.

The centaurin gamma-1 GTPase-like domain functions as an NTPase.

Centaurins are a family of proteins that contain GTPase-activating protein domains, with the gamma family members containing in addition a GTPase-like domain. Centaurins reside mainly in the nucleus and are known to activate phosphoinositide 3-kinase, a key regulator of cell proliferation, motility and vesicular trafficking. In the present study, using X-ray structural analysis, enzymatic assays and nucleotide-binding studies, we show that, for CENTG1 (centaurin gamma-1) the GTPase-like domain has broader trinucleotide specificity. Alterations within the G4 motif of CENTG1 from the highly conserved NKXD found in typical GTPases to TQDR result in the loss of specificity, a lower affinity for the nucleotides and higher turnover rates. These results indicate that the centaurins could be more accurately classified as NTPases and point to alternative mechanisms of cell signalling control.

Structural basis for protein-protein interactions in the 14-3-3 protein family.

The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein-protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.

Phospholipid requirement and pH optimum for the in vitro enzymatic activity of the E. coli P-type ATPase ZntA.

Detergent solubilization and purification of the E. coli heavy metal P-type ATPase ZntA yields an enzyme with reduced hydrolytic activity in vitro. Here, it is shown that the in vitro hydrolytic activity of detergent solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. The protein-lipid interaction of ZntA was characterized by enzyme-coupled ATPase assays and fluorescence spectroscopy. Among the most abundant naturally occurring phospholipids, only phosphatidyl-glycerol lipids (PG) enhance the in vitro enzymatic ATPase activity of ZntA. Re-lipidation of detergent purified ZntA with 1,2-dioleoylphosphatidyl-glycerol (DOPG) increases the ATPase activity four-fold compared to the purified state. All other E. coli phospholipids fail to activate the ATPase. Among the phosphatidyl-glycerol family, highest activity was observed for 1,2-dioleoyl-PG followed by 1,2-dimyristoyl-PG, 1,2-dipalmitoyl-PG and 1,2-distearoyl-PG. Increasing intrinsic Trp fluorescence quantum yield upon relipidation of ZntA was used to determine a pH maximum for lipid binding at pH 6.7. The pH dependence of the lipid binding was confirmed by pH-dependent ATPase assays showing maximum activity at pH 6.7. The biophysical characterization of detergent solubilized membrane proteins crucially relies on the conformational stability and functional integrity of the protein under investigation. The present study describes how the E. coli ZntA P-type ATPase can be stabilized and functionally activated in a detergent solubilized system.